3D printing porous hydrogel based on water-water emulsion as well as preparation method and application of 3D printing porous hydrogel

Abstract

The invention relates to 3D printing porous hydrogel based on a water-water emulsion as well as a preparation method and application of the 3D printing porous hydrogel, and relates to the technical field of biomedicine. The hydrogel is formed by photocuring crosslinking of a hydrogel precursor, the hydrogel precursor comprises a continuous phase medium and a dispersed phase medium, and the volume ratio of the continuous phase medium to the dispersed phase medium is 1: (2-5). The hydrogel provided by the invention breaks through the conventional volume ratio of a continuous phase medium to a dispersed phase medium, and breaks through the volume ratio limitation brought by a traditional water-water two-phase emulsion; the photocuring forming performance, the 3D printing forming performance, the swelling performance and the mechanical performance of the hydrogel are superior to those of existing hydrogel prepared from a high-volume continuous phase medium, so that a new technical system is provided for preparing hydrogel with higher porosity and connectivity.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a 3D-printed porous hydrogel based on a water-water emulsion, its preparation method, and its application. Background Technology

[0002] A water-water emulsion is an emulsion system formed by mixing aqueous solutions of two different water-soluble polymers. The aqueous solutions of these two polymers are miscible at low concentrations, but when the concentration exceeds a certain level, they will separate due to thermodynamic incompatibility, thus forming a water-water emulsion. One phase is the dispersed phase, and the other phase is the continuous phase.

[0003] 3D bioprinting technology refers to the technology that can manufacture specific shapes by shaping and layering bioprinters, bio-inks, cells, growth factors, etc., according to the user's desired shape. In recent years, the use of bioprinting technology to construct precisely controllable three-dimensional scaffolds has become a research hotspot. Bioprinting is a novel additive manufacturing technology that uses biomaterials (such as hydrogels) and biological units (cells, DNA, proteins, etc.) to create personalized biofunctional structures based on biomimetic morphology, biological function, and cell growth microenvironment. Compared with traditional cell-free 3D printing methods, cell-carrying bioprinting can precisely control the density and distribution of cells, constructing bioactive scaffolds that mimic the structure and function of natural tissues.

[0004] Using water-water emulsions as bio-inks for bio-3D printing allows for the creation of porous hydrogel structures after printing by immersion in physiological saline or culture medium. This avoids the limitations of traditional methods using organic materials or subsequent freezing for pore creation, which cannot directly print cells. However, the volume of the continuous phase medium often needs to be greater than or equal to that of the dispersed phase for the resulting water-water emulsion to have a continuous structure. Furthermore, cross-linking of the continuous phase is necessary to form a stable hydrogel scaffold. Conversely, when the volume of the continuous phase is smaller than that of the dispersed phase, an inversion occurs, with the continuous phase becoming the "dispersed phase," while the original dispersed phase becomes the new "continuous phase," making bio-3D printing impossible. Currently, there are no effective methods to further improve the porosity and connectivity of the dispersed phase in bio-3D printed hydrogels. Summary of the Invention

[0005] The purpose of this invention is to address the above-mentioned problems by providing a 3D-printed porous hydrogel based on a water-water emulsion, its preparation method, and its application.

[0006] The first aspect of this invention provides a 3D-printed porous hydrogel based on an water-water emulsion, employing the following technical solution:

[0007] A 3D-printed porous hydrogel based on a water-water emulsion is formed by photocuring and crosslinking a hydrogel precursor. The hydrogel precursor includes a continuous phase medium and a dispersed phase medium, and the volume ratio of the continuous phase medium to the dispersed phase medium is 1:(2-5).

[0008] By adopting the above technical solution, the hydrogel provided by this method breaks through the conventional volume ratio of continuous phase medium and dispersed phase medium, and breaks the volume ratio limitation brought about by traditional water-water two-phase emulsion. The prepared hydrogel has multi-level pore size, and the photocuring molding performance, 3D printing molding performance, swelling performance and mechanical properties of the hydrogel are all better than those of existing hydrogels prepared by high volume continuous phase medium, thus providing a new technical system for preparing hydrogels with higher porosity and connectivity.

[0009] Preferably, the continuous phase medium comprises methacrylamide gelatin (GelMA), gelatin, photoinitiator one, and solvent, wherein the concentration of methacrylamide gelatin is 5% to 20% (w / v), the concentration of gelatin is 0% to 10% (w / v), and the concentration of photoinitiator one is 0.1% to 1% (w / v).

[0010] Preferably, in the continuous phase medium, the concentration of methacrylamide gelatin is 12% (w / v), the concentration of gelatin is 3% (w / v), and the concentration of photoinitiator is 0.5% (w / v).

[0011] Preferably, the dispersed phase medium comprises dextran, photoinitiator II, and solvent, wherein the concentration of dextran is 1%-15% (w / v) and the concentration of photoinitiator II is 0.1%-1% (w / v).

[0012] Preferably, the concentration of dextran in the dispersed phase medium is 5% (w / v), and the concentration of photoinitiator II is 0.5% (w / v).

[0013] Preferably, the solvents in both the continuous phase medium and the dispersed phase medium are phosphate-buffered saline (PBS) at a standard concentration (1*).

[0014] By adopting the above technical solution and adjusting the amount and concentration of each component in the hydrogel precursor, a 3D printed porous hydrogel with better performance can be obtained.

[0015] A second aspect of the present invention provides a method for preparing the above-mentioned water-water emulsion-based 3D-printed porous hydrogel, employing the following technical solution:

[0016] A method for preparing 3D-printed porous hydrogels based on water-water emulsions includes the following steps:

[0017] S1. Preparation of continuous phase medium: Dissolve methacrylamide gelatin, gelatin and photoinitiator in solvent, heat to 37-60℃ and stir to aid dissolution, stir to dissolve in the dark to obtain continuous phase medium, and store the continuous phase medium temporarily at 37℃ in the dark.

[0018] S2. Preparation of the dispersed phase medium: Dissolve dextran and photoinitiator II in a solvent, heat to 37-60℃ and stir to aid dissolution, and obtain the dispersed phase medium. Store the dispersed phase medium temporarily at 4℃ in the dark.

[0019] S3. Preparation of hydrogel precursor: The continuous phase medium obtained in S1 is added to the dispersed phase medium obtained in S2 and mixed in the dark at 0-15℃. After mixing, the mixture is placed at 4℃ for complete physical solidification to obtain the hydrogel precursor.

[0020] S4. Preparation of 3D printed porous hydrogel: The hydrogel precursor obtained in S3 was used as bio-ink, and the 3D printed porous hydrogel was obtained after photocuring and crosslinking.

[0021] By adopting the above technical solution, GelMA and gelatin are partially hydrolyzed collagen products. Their molecular chains can partially restore the triple helix conformation (THS) of natural collagen at low temperatures. The methacryloyl groups of GelMA further stabilize this structure through photocrosslinking. Utilizing the characteristic of GelMA and gelatin mixed solution to form a triple helix structure through self-assembly at low temperatures, a continuous phase medium at 37°C and a dispersed phase medium at 4°C are mixed at low temperatures. After the triple helix structure is restored, the elasticity and deformation resistance of the hydrogel are significantly improved, making it suitable for 3D printing microstructure manufacturing. At the same time, GelMA can simulate and provide a biomimetic microenvironment for cells, promoting directional growth. Combined with dextran solution, it forms multi-level interconnected pores, thereby improving the various properties of the hydrogel.

[0022] Preferably, in the above preparation method technical solution:

[0023] S1. Preparation of continuous phase medium: Dissolve methacrylamide gelatin, gelatin and photoinitiator in solvent, heat to 60°C and stir to aid dissolution, stir to dissolve in the dark to obtain continuous phase medium, and store the continuous phase medium temporarily at 37°C in the dark.

[0024] S2. Preparation of dispersed phase medium: Dissolve dextran and photoinitiator II in solvent, heat to 60°C and stir to aid dissolution, stir to dissolve in the dark to obtain dispersed phase medium, and store the dispersed phase medium temporarily at 4°C in the dark.

[0025] S3. Preparation of hydrogel precursor: The continuous phase medium obtained in S1 is added to the dispersed phase medium obtained in S2 and mixed in the dark at 4°C. After mixing, the mixture is placed at 4°C for complete physical solidification to obtain the hydrogel precursor.

[0026] S4. Preparation of 3D Printing Porous Hydrogel: The hydrogel precursor obtained in S3 was used as the bio-ink, and a light intensity of 25 mW / cm² was used. 2 3D printed porous hydrogels are obtained by curing and crosslinking with light of wavelength 405nm or by using a volumetric 3D printer.

[0027] Both photoinitiator one in S1 and photoinitiator two in S2 are LAP.

[0028] By adopting the above technical solution, the parameters in the preparation method are optimized to improve the performance of 3D printed porous hydrogels.

[0029] A third aspect of the present invention provides a 3D printed scaffold, wherein the raw materials for preparing the 3D printed scaffold include the above-mentioned 3D printed porous hydrogel and biological units, wherein the biological units include any one of cells, DNA, and proteins.

[0030] A fourth aspect of the present invention provides the application of the above-described 3D-printed scaffold in the field of soft tissue repair.

[0031] In summary, this application includes at least one of the following beneficial technical effects:

[0032] 1. The hydrogel provided in this application breaks through the conventional volume ratio of continuous phase medium and dispersed phase medium, and breaks the volume ratio limitation brought about by traditional water-water two-phase emulsion. The prepared hydrogel has multi-level pore size, and the two-dimensional photocuring molding performance, 3D printing molding performance, swelling performance and mechanical properties of the hydrogel are all superior to those of existing hydrogels prepared by high-volume continuous phase medium, thus providing a new technical system for preparing hydrogels with higher porosity and connectivity.

[0033] 2. Utilizing the characteristic of GelMA and gelatin mixtures self-assembling to form a triple helix structure at low temperatures, a continuous phase medium at 37°C and a dispersed phase medium at 4°C are mixed at low temperatures. After the triple helix structure is restored, the elasticity and deformation resistance of the hydrogel are significantly improved, making it suitable for 3D printing microstructure manufacturing. The natural ECM properties of gelatin combined with the crosslinking properties of GelMA provide a biomimetic microenvironment for cells, promoting directional growth. In conjunction with dextran solution, a GelMA-dextran double network structure is formed, creating multi-level interconnected pores, thereby improving the various properties of the hydrogel.

[0034] 3. The hydrogel designed in this invention is formed by physical and chemical dual-network cross-linking; the chemical cross-linking network provides stable mechanical properties, while the physical cross-linking network between GelMA molecules can rebuild bonds after absorbing energy, enhancing its toughness. At the same time, the presence of the triple helix structure further strengthens the GelMA hydrogel. In terms of structure, due to the increased volume ratio of dextran, more continuous dextran phase is formed, further increasing the proportion of interconnected pores. Thus, when the hydrogel is subjected to deformation, water molecules are more easily expelled, increasing the deformation capacity.

[0035] 4. The hydrogel scaffold prepared by mixing the 3D printed porous hydrogel provided by this method with biological units was applied to biological soft tissue repair. It was found that it is more conducive to the survival, proliferation and differentiation of chondrocytes in vivo, thereby accelerating the formation of cartilage tissue. This proves that the hydrogel provided by this method can be used as a source of 3D bioprinting materials and has broad prospects in the field of biological soft tissue repair. Attached Figure Description

[0036] Figure 1 These are the molding performance results of the LSS hydrogels prepared in Examples 1-16 of this application, wherein... Figure 1 Figure a shows the two-dimensional photocuring performance results. Figure 1 b is a graph showing the 3D molding performance results;

[0037] Figure 2 This is a structural characterization diagram of the LSS hydrogel prepared in Example 2 of this application;

[0038] Figure 3 This is a pore size analysis diagram of the LSS hydrogel prepared in Example 2 of this application, wherein, Figure 3 a is an pore size analysis diagram obtained from the SEM image. Figure 3 b is the aperture analysis diagram obtained from the laser confocal image;

[0039] Figure 4 This is a comparison chart of the swelling properties of the LSS hydrogel prepared in Example 2 of this application and the MSN hydrogel prepared in Comparative Examples 1-3;

[0040] Figure 5 This is a photograph of the LSS hydrogel prepared in Example 2 of this application, wherein... Figure 5 Image a shows an LSS hydrogel that recovers its original structure after compression, bending, and torsion. Figure 5 b is a photograph of the LSS hydrogel being stretched and knotted.

[0041] Figure 6 The graph shows the mechanical property test results of the LSS hydrogel prepared in Example 2 of this application, the MSN hydrogel prepared in Comparative Example 1, and the NDN hydrogel prepared in Comparative Example 4. Figure 6 a is the stress-strain curve of MSN, NDN, and LSS hydrogels. Figure 6 b shows the stress-strain curves of the LSS hydrogel under loading and unloading at 20%, 40%, 60%, and 80% loading rates. Figure 6 c is a statistical graph of the maximum compressibility of MSN, NDN, and LSS hydrogels. Figure 6 d is a statistical graph of the maximum compressive strength of MSN, NDN, and LSS hydrogels. Figure 6 e is a statistical graph of the toughness of MSN, NDN and LSS hydrogels;

[0042] Figure 7 This is a schematic diagram of the operation of implanting the hydrogel scaffold into nude mice in Application Examples 1-3 of this application;

[0043] Figure 8 These are performance test diagrams of the hydrogel scaffold implanted in the application examples of this application, wherein... Figure 8 Image a shows the H&E, safranin-O, type II collagen immunohistochemistry, elastin, and toluidine blue staining images of different hydrogel samples 60 days after implantation. Figure 8 bg represents the quantitative statistical results of toluidine blue, safranin-O, elastin, type II collagen immunohistochemical, stress-strain, and Young's modulus of hydrogel samples 60 days after implantation.

[0044] Figure 9 These are test images of the 3D printing performance of the LSS hydrogel provided in this application, in which... Figure 9 A is the pre-printing design structure diagram. Figure 9 b is a picture of the printed hydrogel scaffold;

[0045] Figure 10 This is a staining image of the LSS hydrogel printing performance test provided in this application. Detailed Implementation

[0046] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Modifications or equivalent substitutions made by those skilled in the art based on their understanding of the technical solutions of this invention, without departing from the spirit and scope of the invention, should be covered within the protection scope of this invention.

[0047] The reagent information used in the following examples is as follows. All reagents and chemicals were used as is without further processing. Other specific conditions not specified were performed according to standard conditions or the manufacturer's recommendations. Reagents or instruments whose manufacturers are not specified were all commercially available products.

[0048] Reagent source:

[0049] Methacrylamide gelatin (GelMA): It is prepared by compounding methacrylic anhydride and gelatin. The preparation method is based on the journal DOI:10.1038 / srep31036, or commercially available finished products can be used.

[0050] Gelatin: Type A (pigskin), purchased from Sigma;

[0051] Dextran: molecular weight 500,000, purchased from J&K Scientifi;

[0052] Photoinitiator 1 and Photoinitiator 2: Both use LAP, CAS: 85073-19-4; the rest are purchased from commercially available sources unless otherwise specified.

[0053] The abbreviations and meanings of reagents used in this application are as follows:

[0054] Methacrylamide gelatin (GelMA), gelatin, dextran, lithium phenyl-2,4,6-trimethylbenzoylphosphonite (LAP), phosphate buffer (PBS), low-temperature self-assembled superporous hydrogel (LSS hydrogel), nanoporous double-network hydrogel (NDN hydrogel), microporous single-network hydrogel (MSN hydrogel), and other hydrogels not specifically mentioned are subject to industry-standard understanding.

[0055] This application provides a 3D-printed porous hydrogel based on a water-water emulsion, which is formed by photocuring and crosslinking a hydrogel precursor, the hydrogel precursor including a continuous phase medium and a dispersed phase medium.

[0056] The continuous phase medium includes methacrylamide gelatin (GelMA), gelatin, photoinitiator 1, and solvent. The concentration of methacrylamide gelatin is 5% to 20% (w / v), preferably 12% (w / v); the concentration of gelatin is 0% to 10% (w / v), preferably 3% (w / v); and photoinitiator 1 is LAP, with a concentration of 0.1% to 1% (w / v), preferably 0.5% (w / v).

[0057] The dispersed phase medium includes dextran, photoinitiator II, and solvent. The concentration of dextran is 1%-15% (w / v), preferably 5% (w / v). The photoinitiator II is LAP, with a concentration of 0.1% to 1% (w / v), preferably 0.5% (w / v). The solvent for both the continuous phase medium and the dispersed phase medium is phosphate-buffered saline (1*PBS) of standard concentration.

[0058] I. Implementation Examples

[0059] Example 1

[0060] A method for preparing 3D-printed porous hydrogels based on water-water emulsions includes the following steps:

[0061] S1. Preparation of continuous phase medium: Dissolve methacrylamide gelatin, gelatin and photoinitiator-LAP in 1*PBS solution, heat to 37°C and stir to dissolve in the dark to obtain continuous phase medium. The concentrations of each component in the continuous phase medium are: methacrylamide gelatin 5% (w / v), gelatin 0% (w / v), LAP 0.1% (w / v). Store the continuous phase medium temporarily at 37°C in the dark.

[0062] S2. Preparation of the dispersed phase medium: Dissolve dextran and photoinitiator LAP in 1*PBS solution, heat to 37°C and stir in the dark to dissolve, to obtain the dispersed phase medium. The concentration of dextran in the dispersed phase medium is 1% (w / v) and LAP is 0.1% (w / v). Store the dispersed phase medium in the dark at 4°C.

[0063] S3. Preparation of hydrogel precursor: The continuous phase medium obtained in S1 is added to the dispersed phase medium obtained in S2. The volume ratio of the continuous phase medium to the dispersed phase medium is 1:2. The mixture is mixed in the dark at 0°C. After mixing, it is placed at 4°C for complete physical solidification to obtain the hydrogel precursor.

[0064] S4. Preparation of 3D Printing Porous Hydrogel: The hydrogel precursor obtained in S3 was used as the bio-ink, and a light intensity of 25 mW / cm² was used. 2 The 3D printed porous hydrogel was cured and crosslinked by irradiation with light of wavelength 405nm for 10s.

[0065] After the hydrogel has cured and crosslinked, it is necessary to remove dextran and gelatin. The removal principle is the same as that in the prior art. The operation steps refer to the common removal methods and steps in the prior art, which will not be repeated in this application. The general principle is as follows: After the photocuring and crosslinking is completed, the hydrogel is soaked in an aqueous solution or culture medium and removed by diffusion through the concentration gradient of the material. Depending on the differences in material concentration and degree of crosslinking, the removal is achieved by soaking for 24 hours.

[0066] The 3D-printed porous hydrogel based on water-water emulsion prepared according to the preparation method provided in this embodiment is designated as LSS-1.

[0067] Example 2

[0068] A method for preparing 3D-printed porous hydrogels based on water-water emulsions includes the following steps:

[0069] S1. Preparation of continuous phase medium: Dissolve methacrylamide gelatin, gelatin and photoinitiator-LAP in 1*PBS solution, heat to 37°C and stir to dissolve in the dark to obtain continuous phase medium. The concentrations of each component in the continuous phase medium are: methacrylamide gelatin 12% (w / v), gelatin 3% (w / v), LAP 0.5% (w / v). Store the continuous phase medium temporarily at 37°C in the dark.

[0070] S2. Preparation of the dispersed phase medium: Dissolve dextran and photoinitiator LAP in 1*PBS solution, heat to 37°C and stir in the dark to dissolve, to obtain the dispersed phase medium. The concentration of dextran in the dispersed phase medium is 5% (w / v) and LAP is 0.5% (w / v). Store the dispersed phase medium in the dark at 4°C.

[0071] S3. Preparation of hydrogel precursor: The continuous phase medium obtained in S1 is added to the dispersed phase medium obtained in S2. The volume ratio of the continuous phase medium to the dispersed phase medium is 1:2. After mixing, the mixture is placed at 4°C for complete physical solidification to obtain the hydrogel precursor.

[0072] S4. Preparation of 3D Printing Porous Hydrogel: The hydrogel precursor obtained in S3 was used as the bio-ink, and a light intensity of 25 mW / cm² was used. 2 The 3D printed porous hydrogel was cured and crosslinked by irradiation with light of wavelength 405nm for 10s.

[0073] The 3D-printed porous hydrogel based on water-water emulsion prepared according to the preparation method provided in this embodiment is denoted as LSS-2.

[0074] Example 3

[0075] A method for preparing 3D-printed porous hydrogels based on water-water emulsions includes the following steps:

[0076] S1. Preparation of continuous phase medium: Dissolve methacrylamide gelatin, gelatin and photoinitiator-LAP in 1*PBS solution, heat to 37°C and stir to dissolve in the dark to obtain continuous phase medium. The concentrations of each component in the continuous phase medium are: methacrylamide gelatin 20% (w / v), gelatin 10% (w / v), LAP 1% (w / v). Store the continuous phase medium temporarily at 37°C in the dark.

[0077] S2. Preparation of dispersed phase medium: Dissolve dextran and photoinitiator LAP in 1*PBS solution, heat to 37℃ and stir to dissolve in the dark to obtain dispersed phase medium. The concentration of dextran in the dispersed phase medium is 10% (w / v) and LAP is 1% (w / v). Store the dispersed phase medium in the dark at 4℃.

[0078] S3. Preparation of hydrogel precursor: The continuous phase medium obtained in S1 is added to the dispersed phase medium obtained in S2. The volume ratio of the continuous phase medium to the dispersed phase medium is 1:2. The mixture is mixed in the dark at 15°C. After mixing, it is placed at 4°C for complete physical solidification to obtain the hydrogel precursor.

[0079] S4. Preparation of 3D Printing Porous Hydrogel: The hydrogel precursor obtained in S3 was used as the bio-ink, and a light intensity of 25 mW / cm² was used. 2 The 3D printed porous hydrogel was cured and crosslinked by irradiation with light of wavelength 405nm for 10s.

[0080] The 3D-printed porous hydrogel based on water-water emulsion prepared according to the preparation method provided in this embodiment is designated as LSS-3.

[0081] Example 4

[0082] A method for preparing a 3D-printed porous hydrogel based on an aqueous emulsion, differing from Example 2 in that the concentration of dextran in the dispersed phase medium is 15% (w / v), and the 3D-printed porous hydrogel based on an aqueous emulsion prepared according to the method provided in this example is designated as LSS-4.

[0083] Example 5

[0084] A method for preparing a 3D-printed porous hydrogel based on an aqueous emulsion differs from Example 2 in that the concentration of dextran in the dispersed phase medium is 1% (w / v), and the volume ratio of the dispersed phase medium to the continuous phase medium is 1:3. The 3D-printed porous hydrogel based on an aqueous emulsion prepared according to the method provided in this example is designated as LSS-5.

[0085] Example 6

[0086] A method for preparing a 3D-printed porous hydrogel based on an water-water emulsion, which differs from Example 2 in that the volume ratio of the dispersed phase medium to the continuous phase medium is 1:3, and the 3D-printed porous hydrogel based on an water-water emulsion prepared according to the method provided in this example is designated as LSS-6.

[0087] Example 7

[0088] A method for preparing a 3D-printed porous hydrogel based on an aqueous emulsion differs from Example 2 in that the concentration of dextran in the dispersed phase medium is 10% (w / v), and the volume ratio of the dispersed phase medium to the continuous phase medium is 1:3. The 3D-printed porous hydrogel based on an aqueous emulsion prepared according to the method provided in this example is designated as LSS-7.

[0089] Example 8

[0090] A method for preparing a 3D-printed porous hydrogel based on an aqueous emulsion differs from Example 2 in that the concentration of dextran in the dispersed phase medium is 15% (w / v), and the volume ratio of the dispersed phase medium to the continuous phase medium is 1:3. The 3D-printed porous hydrogel based on an aqueous emulsion prepared according to the method provided in this example is designated as LSS-8.

[0091] Example 9

[0092] A method for preparing a 3D-printed porous hydrogel based on an aqueous emulsion differs from Example 2 in that the concentration of dextran in the dispersed phase medium is 1% (w / v), and the volume ratio of the dispersed phase medium to the continuous phase medium is 1:4. The 3D-printed porous hydrogel based on an aqueous emulsion prepared according to the method provided in this example is designated as LSS-9.

[0093] Example 10

[0094] A method for preparing a 3D-printed porous hydrogel based on an water-water emulsion, which differs from Example 2 in that the volume ratio of the dispersed phase medium to the continuous phase medium is 1:4, and the 3D-printed porous hydrogel based on an water-water emulsion prepared according to the method provided in this example is designated as LSS-10.

[0095] Example 11

[0096] A method for preparing a 3D-printed porous hydrogel based on an aqueous emulsion differs from Example 2 in that the concentration of dextran in the dispersed phase medium is 10% (w / v), and the volume ratio of the dispersed phase medium to the continuous phase medium is 1:4. The 3D-printed porous hydrogel based on an aqueous emulsion prepared according to the method provided in this example is designated as LSS-11.

[0097] Example 12

[0098] A method for preparing a 3D-printed porous hydrogel based on an aqueous emulsion differs from Example 2 in that the concentration of dextran in the dispersed phase medium is 15% (w / v), and the volume ratio of the dispersed phase medium to the continuous phase medium is 1:4. The 3D-printed porous hydrogel based on an aqueous emulsion prepared according to the method provided in this example is designated as LSS-12.

[0099] Example 13

[0100] A method for preparing a 3D-printed porous hydrogel based on an aqueous emulsion differs from Example 2 in that the concentration of dextran in the dispersed phase medium is 1% (w / v), and the volume ratio of the dispersed phase medium to the continuous phase medium is 1:5. The 3D-printed porous hydrogel based on an aqueous emulsion prepared according to the method provided in this example is designated as LSS-13.

[0101] Example 14

[0102] A method for preparing a 3D-printed porous hydrogel based on an water-water emulsion, which differs from Example 2 in that the volume ratio of the dispersed phase medium to the continuous phase medium is 1:5, and the 3D-printed porous hydrogel based on an water-water emulsion prepared according to the method provided in this example is designated as LSS-14.

[0103] Example 15

[0104] A method for preparing a 3D-printed porous hydrogel based on an aqueous emulsion differs from Example 2 in that the concentration of dextran in the dispersed phase medium is 10% (w / v), and the volume ratio of the dispersed phase medium to the continuous phase medium is 1:5. The 3D-printed porous hydrogel based on an aqueous emulsion prepared according to the method provided in this example is designated as LSS-15.

[0105] Example 16

[0106] A method for preparing a 3D-printed porous hydrogel based on an aqueous emulsion differs from Example 2 in that the concentration of dextran in the dispersed phase medium is 15% (w / v), and the volume ratio of the dispersed phase medium to the continuous phase medium is 1:5. The 3D-printed porous hydrogel based on an aqueous emulsion prepared according to the method provided in this example is designated as LSS-16.

[0107] II. Comparative Example

[0108] Comparative Example 1

[0109] This comparative example provides an MSN hydrogel, the preparation method of which differs from that of Example 2 in that: the concentration of dextran in the dispersed phase medium is 10% (w / v), the dispersed phase medium is temporarily stored at 37°C in the dark, and the continuous phase medium is added to the dispersed phase medium at a volume ratio of 2:1. After mixing at 37°C, a hydrogel precursor is obtained; this hydrogel precursor is used as a bio-ink, with a light intensity of 25 mW / cm². 2 The MSN hydrogel was cured and crosslinked by irradiation with light of wavelength 405nm for 10s, and was denoted as MSN-1.

[0110] Comparative Example 2

[0111] This comparative example provides an MSN hydrogel, the preparation method of which differs from Comparative Example 1 in that the volume ratio of the continuous phase medium to the dispersed phase medium is 3:1; this hydrogel precursor is used as a bio-ink, and a light intensity of 25 mW / cm² is employed. 2 The MSN hydrogel was cured and crosslinked by irradiation with light of wavelength 405nm for 10s, and was denoted as MSN-2.

[0112] Comparative Example 3

[0113] This comparative example provides an MSN hydrogel, the preparation method of which differs from that of Comparative Example 1 in that the volume ratio of the continuous phase medium to the dispersed phase medium is 4:1; this hydrogel precursor is used as a bio-ink, and a light intensity of 25 mW / cm² is employed. 2 The MSN hydrogel was cured and crosslinked by irradiation with light of wavelength 405nm for 10s, and was denoted as MSN-3.

[0114] Comparative Example 4

[0115] This comparative example provides an NDN hydrogel, the preparation method of which differs from that of Example 2 in that: there is no dispersed phase medium in the precursor solution, the continuous phase medium is prepared and placed at 4°C for physical gelation, and after gelation, it is printed by a volumetric 3D printer to obtain the NDN hydrogel.

[0116] III. Hydrogel Performance Testing Experiments and Results

[0117] 1. Molding performance test

[0118] The LSS hydrogels prepared in Examples 1-16 were subjected to two-dimensional photocuring and 3D molding performance tests, respectively. The test methods are as follows:

[0119] Test results are as follows Figure 1 As shown, where Figure 1 a is a statistical chart of two-dimensional photocurable molding performance. Figure 1Figure b shows the statistical chart of 3D printing molding performance, where the horizontal axis GelMA:Dextran represents the volume ratio between the continuous phase medium containing GelMA and the dispersed phase medium containing Dextran. As shown in the figure, under 2D printing conditions, when the volume ratio of GelMA:Dextran solution is between 1:2 and 1:5, the molding performance decreases with increasing Dextran concentration; it also gradually decreases with increasing Dextran volume ratio. The same pattern applies to 3D printing. Furthermore, the molding range of LSS hydrogel is further narrowed in 3D printing. Based on the test results, Example 2 exhibits the best molding performance; therefore, Example 2 is the optimal example. To obtain better mechanical properties and cell activity, we selected GelMA:Dextan = 1:2 and a Dextran concentration of 5% (w / v) for the preparation of this material system.

[0120] 2. Structural Characterization

[0121] The LSS-2 hydrogel prepared in Example 2 was structurally characterized, and SEM images and confocal images at different magnifications are shown below. Figure 2 As shown in the SEM image, the LSS hydrogel has an interconnected multi-level structure. The multi-level dispersed pore structure is also an important factor in achieving the good mechanical properties of the hydrogel. The microstructure of the hydrogel in the water-containing state was analyzed by laser confocal microscopy, and the results were consistent with the SEM image.

[0122] 3. Pore structure analysis

[0123] Further statistical analysis was performed on the pore structure of the LSS-2 hydrogel prepared in Example 2. One hundred pore structures were counted, and then plotted using Origin software. The results are as follows: Figure 3 As shown, where, Figure 3 a is an pore size analysis diagram obtained from the SEM image. Figure 3 b is the aperture analysis diagram obtained from the laser confocal image.

[0124] As shown in the figure, the pore size distribution of the LSS hydrogel is mainly in two ranges: macropores with a diameter of about 125 μm, and the supporting structure that constitutes the macropores is composed of micropores of about 1.6 μm. The statistical results of the laser confocal microscope images are similar to those of the SEM images.

[0125] 4. Swelling performance test

[0126] The swelling rate of LSS-2 prepared in Example 2 and MSN hydrogels prepared in Comparative Examples 1-3 were tested respectively. The test method is as follows: the mass of the formed hydrogel was weighed as M0, and then the hydrogel was immersed in 1*PBS and left to stand at 37°C for 24 hours. The mass was then weighed again as M1.

[0127] Swelling rate = (M1 - M0) / M0 * 100%

[0128] Test results are as follows Figure 4 As shown in the figure, the swelling rate of MSN hydrogel gradually increases with the increase of the volume ratio of the pore-forming agent dextran; however, even with the highest volume ratio of the pore-forming agent, the swelling rate of LSS hydrogel is significantly lower than that of MSN hydrogel, and the swelling rate of LSS is negative, indicating that LSS hydrogel shrinks. This phenomenon may be due to the double cross-linked network structure of LSS hydrogel. The shrinkage phenomenon of LSS hydrogel can effectively improve printing accuracy and printing fidelity.

[0129] 5. Mechanical property testing

[0130] The LSS hydrogel prepared in Example 2, the MSN hydrogel prepared in Comparative Example 1, and the NDN hydrogel prepared in Comparative Example 4 were printed with the same structure (columnar structure) using a volumetric 3D printer. The mechanical properties of the formed hydrogels were tested under the following conditions: compression rate 1 mm / min and sampling frequency 3000 points / min.

[0131] Test results are as follows Figure 5 and Figure 6 As shown, where Figure 5 Image a shows an LSS hydrogel that recovers its original structure after compression, bending, and torsion. Figure 5 b is a photograph of the LSS hydrogel being stretched and knotted. Figure 6 a represents the stress-strain curves of MSN, NDN, and LSS hydrogels. Figure 6 b represents the stress-strain curves of the LSS hydrogel under loading and unloading at 20%, 40%, 60%, and 80% loading rates. Figure 6 c is a statistical chart of the maximum compressibility of MSN, NDN, and LSS hydrogels. Figure 6 d is a statistical graph of the maximum compressive strength of MSN, NDN, and LSS hydrogels. Figure 6 e is a statistical graph of the toughness of MSN, NDN, and LSS hydrogels.

[0132] Traditionally, single-component GelMA hydrogels often exhibit high brittleness and are prone to fracture under external mechanical stimuli. Figure 5 and Figure 6The study revealed that the prepared LSS possesses excellent mechanical properties and can undergo various mechanical deformations. Under the compression mechanics model, LSS does not exhibit longitudinal mechanical imbalance even with 80% compression deformation, while MSN and NDN hydrogels exhibit mechanical imbalance at approximately 30% and 40% deformation, respectively. This result further demonstrates that LSS hydrogel has greater toughness.

[0133] IV. Application Examples

[0134] Application Example 1

[0135] This application example provides a 3D printed scaffold. The raw materials for preparing the 3D printed scaffold include the 3D printed porous hydrogel provided in Example 2 and biological units. The biological units include any one of cells, DNA, and proteins. In this application example, the biological unit is chondrocytes.

[0136] Chondrocyte source: Rabbit ear cartilage, generation P3-5, extracted in-house according to the laboratory method of the PLA Army Characteristic Medical Center.

[0137] The 3D printed scaffold is prepared as follows: A continuous phase medium is prepared according to the preparation method in S1 of Example 2. After the continuous phase medium is prepared, it is placed at 37°C. Rabbit ear chondrocytes are then added and mixed. The cell density here is 1*102 7 The mixture was obtained, and the dispersed phase medium was prepared according to the preparation method of S3 in Example 2. The mixture and the dispersed phase medium were mixed at 4°C at a volume ratio of 1:2 according to the preparation method of S3 in Example 2. After mixing, the mixture was cured and crosslinked according to the method of S4. After curing and crosslinking, the gelatin and dextran were removed by immersion in an aqueous solution or culture medium. Depending on the differences in material concentration and degree of crosslinking, the removal was achieved in 24 hours. The prepared scaffold was denoted as LSS hydrogel scaffold.

[0138] Reference Figure 7 The prepared LSS hydrogel scaffold was implanted subcutaneously into the back of nude mice purchased from Huafukang Laboratory Animal Company.

[0139] Application Example 2

[0140] This application example provides an MSN hydrogel scaffold. The raw materials for preparing the MSN hydrogel scaffold include the MSN hydrogel provided in Comparative Example 1 and rabbit ear chondrocytes. The scaffold is prepared according to the preparation method provided in Comparative Example 1. The difference is that after the continuous phase medium is prepared, rabbit ear chondrocytes are added and mixed to form a mixture. Then, the mixture and the dispersed phase medium are mixed at 37°C at a volume ratio of 2:1 according to the preparation method in Comparative Example 1. After mixing, the mixture is cured and crosslinked by irradiation with 405nm wavelength light (light intensity: 25mW / cm2) for 10s to obtain the MSN hydrogel scaffold.

[0141] Reference Figure 7 The prepared MSN hydrogel scaffold was implanted subcutaneously into the back of nude mice.

[0142] Application Example 3

[0143] This application example provides an NDN hydrogel scaffold. The raw materials for preparing the NDN hydrogel scaffold include the NDN hydrogel provided in Comparative Example 4 and rabbit ear chondrocytes. The scaffold is prepared according to the preparation method provided in Comparative Example 4, except that after the continuous phase medium is prepared, rabbit ear chondrocytes are added and mixed. The mixture is placed at 4°C to physically gel, and after gelation, it is cured and crosslinked by irradiation with 405nm wavelength light (light intensity: 25mW / cm2) for 10s to obtain the MSN hydrogel scaffold.

[0144] V. Performance Testing Experiments and Results of Implanted 3D Printed Scaffold

[0145] Test Items and Methods: Performance tests were conducted on the hydrogel scaffolds implanted in test cases 1-3. The cartilage regeneration hydrogel was implanted subcutaneously in nude mice for 60 days, followed by tissue sampling and staining. The specific steps were as follows: After sampling, the tissue was immersed in 4% paraformaldehyde, and then further dehydrated, embedded, sectioned, and stained. Safranin O staining solution was purchased from Solarbio (catalog number: G2540). The specific steps were as follows: After routine dewaxing of paraffin sections to water, the sections were immersed in cartilage staining solution (Safranin O method) for 1 min, washed with distilled water for 1 min; further dehydration was performed with 95% ethanol and anhydrous ethanol respectively; xylene was used for clearing, and optical resin was used for mounting. Type II collagen was stained using immunohistochemistry, and the antibody used for staining was from Bioss, with an antibody ratio of 1:200.

[0146] Test results are as follows Figure 8 a and Figure 8 As shown in (bg), Figure 8 Image a shows H&E, safranin-O, type II collagen immunohistochemistry, elastin, and toluidine blue staining of different hydrogel samples 60 days after implantation. Figure 8 Figure b shows the quantitative statistical results of toluidine blue in hydrogel samples 60 days after implantation. Figure 8 c is the statistical result of safranin-O quantification in hydrogel samples 60 days after implantation. Figure 8 d is a graph showing the quantitative statistical results of elastin in hydrogel samples 60 days after implantation. Figure 8 Figure e shows the statistical results of immunohistochemical quantification of type II collagen in hydrogel samples 60 days after implantation. Figure 8 f represents the stress-strain diagrams of implanted samples from different groups 60 days after implantation. Figure 8 g is a graph showing the statistical results of Young's modulus of implanted samples from different groups 60 days after implantation.

[0147] Depend on Figure 8Histological staining results showed that, compared to the NDN and MSN groups, the LSS group exhibited more mature new cartilage tissue formation, and the cartilage tissue grew in sheet-like patterns within the interconnecting pores of the hydrogel. In the NDN group, a large number of implanted chondrocytes had died, leaving numerous pores. In the MSN group, although some chondrocytes survived, they were distributed in scattered punctate patterns. These results indicate that the LSS hydrogel provided in this application is more conducive to the survival, proliferation, and differentiation of chondrocytes in vivo, thereby accelerating the formation of cartilage tissue.

[0148] VI. Printing Performance Test

[0149] Following the method provided in Example 2, volumetric 3D printing technology based on digital light processing was used, employing the hydrogel precursor provided in Example 2 as the bio-ink for printing. Figure 9 'a' represents the pre-printing design structure. Figure 9 b shows the printed hydrogel scaffold, demonstrating that LSS hydrogel can rapidly construct different structures with minimal deviation from the ideal design, and can match and conform to existing soft tissue structures. Following the method provided in Application Example 1, cells were loaded and 3D printed, and staining experiments were performed. The results are as follows... Figure 10 As shown in the figure, cells exhibit high cell viability in LSS hydrogel and can remodel the cytoskeleton. This result indicates that LSS hydrogel has good printing performance and can be used as a source of bio-3D printing materials.

[0150] In summary, the hydrogel provided in this application breaks through the conventional volume ratio of continuous phase medium and dispersed phase medium, and overcomes the volume ratio limitation brought about by traditional water-water two-phase emulsions. The prepared hydrogel has multi-level pore size, and the two-dimensional photocuring molding performance, 3D printing molding performance, swelling performance and mechanical properties of the hydrogel are all superior to those of existing hydrogels prepared by high-volume continuous phase media. Thus, it provides a new technical system for preparing hydrogels with higher porosity and connectivity.

[0151] The hydrogel scaffold prepared by mixing the 3D-printed porous hydrogel provided by this method with biological units was applied to biological soft tissue repair. It was found that it is more conducive to the survival, proliferation and differentiation of chondrocytes in vivo, thereby accelerating the formation of cartilage tissue. This proves that the hydrogel provided by this method can be used as a source of 3D bioprinting materials and has broad prospects in the field of biological soft tissue repair.

Claims

1. A water-water emulsion based 3D printed porous hydrogel, characterized in that: is prepared by photo-crosslinking a hydrogel precursor, the hydrogel precursor comprising a continuous phase medium and a dispersed phase medium, a volume ratio of the continuous phase medium and the dispersed phase medium being 1:(2-5).

2. The water-water emulsion based 3D printed porous hydrogel according to claim 1, characterized in that: The continuous phase medium comprises methacrylated gelatin (GelMA), gelatin, a photo-initiator I, and a solvent, a concentration of the methacrylated gelatin being 5%-20% (w / v), a concentration of the gelatin being 0%-10% (w / v), and a concentration of the photo-initiator I being 0.1%-1% (w / v).

3. The water-water emulsion based 3D printed porous hydrogel according to claim 2, wherein: In the continuous phase medium, the concentration of the methacrylated gelatin is 12% (w / v), the concentration of the gelatin is 3% (w / v), and the concentration of the photo-initiator I is 0.5% (w / v).

4. The water-water emulsion based 3D printed porous hydrogel according to claim 2, wherein: The dispersed phase medium comprises dextran, a photo-initiator II, and a solvent, a concentration of the dextran being 1%-15% (w / v), and a concentration of the photo-initiator II being 0.1%-1% (w / v).

5. The water-water emulsion based 3D printed porous hydrogel according to claim 4, characterized in that: In the dispersed phase medium, the concentration of the dextran is 5% (w / v), and the concentration of the photo-initiator II is 0.5% (w / v).

6. The water-water emulsion based 3D printed porous hydrogel according to claim 4, wherein: The solvent in the continuous phase medium and the dispersed phase medium is standard concentration (1*) phosphate buffered saline (PBS).

7. Process for the preparation of a water-water emulsion based 3D printed porous hydrogel according to any one of claims 1-6, characterized in that: The method comprises the following steps: S1, configuring a continuous phase medium: dissolving methacrylated gelatin, gelatin, and a photo-initiator I in a solvent, heating to 37-60°C to aid dissolution by stirring, stirring to dissolve in the dark, obtaining the continuous phase medium, and storing the continuous phase medium at 37°C in the dark; S2, configuring a dispersed phase medium: dissolving dextran and a photo-initiator II in a solvent, heating to 37-60°C to aid dissolution by stirring, obtaining the dispersed phase medium, and storing the dispersed phase medium at 4°C in the dark; S3, preparing a hydrogel precursor: adding the continuous phase medium obtained in S1 to the dispersed phase medium obtained in S2, mixing in the dark at 0-15°C, and obtaining the hydrogel precursor after complete physical solidification at 4°C; S4, preparing a 3D-printed porous hydrogel: using the hydrogel precursor prepared in S3 as bio-ink, and obtaining the 3D-printed porous hydrogel after photo-crosslinking.

8. The preparation method according to claim 7, wherein: S1, configuring a continuous phase medium: dissolving methacrylated gelatin, gelatin, and a photo-initiator I in a solvent, heating to 60°C to aid dissolution by stirring, stirring to dissolve in the dark, obtaining the continuous phase medium, and storing the continuous phase medium at 37°C in the dark; S2, configuring a dispersed phase medium: dissolving dextran and a photo-initiator II in a solvent, heating to 60°C to aid dissolution by stirring, stirring to dissolve in the dark, obtaining the dispersed phase medium, and storing the dispersed phase medium at 4°C in the dark; S3, preparing a hydrogel precursor: adding the continuous phase medium obtained in S1 to the dispersed phase medium obtained in S2, mixing in the dark at 4°C, and obtaining the hydrogel precursor after complete physical solidification at 4°C; S4, preparing 3D printing porous hydrogel: using the hydrogel precursor prepared in S3 as a biological ink, using light with an intensity of 25 mW / cm2and a wavelength of 405 nm for curing and crosslinking or using a volume 3D printer for curing and crosslinking to obtain a 3D printing porous hydrogel; Both the photo-initiator one in S1 and the photo-initiator two in S2 are selected from LAP.

9. A 3D printed scaffold, characterized in that: The raw materials for preparing the 3D printing scaffold include the 3D printing porous hydrogel according to any one of claims 1-6 and a biological unit, and the biological unit includes any one of cells, DNA and proteins.

10. Application of the 3D printing scaffold according to claim 9 in the field of soft tissue repair.

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